DESCRIPTION: In the eukaryotic cell, DNA is packaged into chromatin, which regulates its accessibility for fundamental processes such as DNA replication, transcription, and repair. The first level of packaging is the nucleosome and consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of histones H2A, H2B, H3, and H4. While structural studies traditionally depict the nucleosome in a single conformation [8], likely stabilized by the crystal lattice, some evidence revealed the presence of structural dynamism in nucleosomes in solution. The DNA ends were observed to spontaneously undergo rapid and reversible unwrapping from the octamer [2-4, 9]. Additionally, the H2A-H2B dimer was seen to at least partially detach from the (H3-H4)2 tetramer [1, 4]. This conformational dynamism may help regulate the accessibility of DNA to chromatin interacting factors and the composition of the nucleosome may affect its propensity to adopt a more open state. The specific aims of this proposal thus seek to investigate alternative, more open conformations in nucleosomes in solution. The first aim will be to implement strategies to analyze nucleosome conformational dynamics in vitro, including a fluorescence assay, a method to probe the accessibility of buried histone residues at interfaces, and hydrogen- deuterium exchange analysis. The second aim will characterize the effect of nucleosome components, namely histone H2A variants and DNA sequence, on conformational dynamics in vitro. Lastly, the third aim will investigate the propensity of H2A- and H2A.Z- containing yeast nucleosomes to adopt an open conformation in vivo. Altogether, this work will assess the presence, and characterize the nature of nucleosome open states both in vitro and in vivo. The resulting insights may radically alter our view of chromatin regulation and its ramifications on basic cellular processes and the etiology of diseases such as cancer [5-7].